Method of use of peptide inhibitors in cancer treatment
Patent Information
- Authority / Receiving Office
- EP · EP
- Patent Type
- Applications
- Current Assignee / Owner
- ISTANBUL TEKNIK UNIVSI
- Filing Date
- 2024-08-21
- Publication Date
- 2026-07-01
AI Technical Summary
Current cancer treatments face challenges due to acquired drug resistance in cancer cells, which limits the effectiveness of existing inhibitors targeting kinases and underutilizes inhibitors targeting protein-protein interactions.
The method involves synthesizing peptide inhibitors using a FMOC-based solid-phase peptide synthesis approach, specifically targeting the Bag-1/C-Raf interaction, and using derivatives like P2 to enhance cell permeability and effectiveness against resistant Ras-mutant tumors.
This approach disrupts the Bag-1/C-Raf complex, offering therapeutic benefits in treating breast, colon, and resistant Ras-mutant tumors by enhancing treatment efficacy and overcoming drug resistance.
Smart Images
Figure TR2024050975_27022025_PF_FP_ABST
Abstract
Description
[0001] DESCRIPTION
[0002] METHOD OF USE OF PEPTIDE INHIBITORS IN CANCER TREATMENT
[0003] Technical Field
[0004] The invention is related to a method of using peptide inhibitors in cancer treatment targeting bag- 1 -mediated complexes, in particular the bag-l / c-raf complex.
[0005] Prior Art
[0006] In the pharmaceutical and biotechnology sector and in the field of molecular and experimental oncology, peptide inhibitors have the potential to be used therapeutically in cancer treatment with single or combined approaches.
[0007] A large number of inhibitors targeting different pathways are available for use in cancer treatment. However, “acquired drug resistance” occurring in cancer cells during / after treatment neutralises the response of patients to drugs, leading to treatment failure or relapse of the disease. Acquired drug resistance depends on multiple independent mechanisms such as intracellular detoxification, alterations in cellular response, stress tolerance and deregulations in apoptosis signalling pathways. In addition, it has been shown that for effective therapy, blocking cancer-causing signalling pathways at multiple points increases the success of treatment. Accordingly, current approaches to achieve effective therapeutic benefit have focused on pharmaceutical combinations of molecularly targeted therapies. Therefore, the development of inhibitors for new molecular targets is critical for changing and improving treatment strategies.
[0008] Most of the inhibitors used have been developed for kinases with enzymatic activity. These inhibitors usually target the catalytic site in the protein. However, the number of inhibitors targeting the interaction regions of proteins is quite low due to the incompatibility in the three-dimensional structure of most proteins despite their high success potential.
[0009] In the technical field, the Bag-1 peptide which prevents prostate cancer is known. Accordingly, the invention numbered EP2151452 relates to a Bag-1 peptide which prevents prostate cancer. The invention discloses a polypeptide having antitumour properties, a vector encoding said polypeptide, a host cell containing said vector, the use of said polypeptide in the production of a drug for the treatment of cancer in patients, a method for binding said polypeptide to molecular chaperones, and a pharmaceutical composition comprising said polypeptide. However, it does not involve synthesising a new peptide as a derivative of the peptide to confer cell permeable function to the peptides. That is, there is no use of a peptide and its derivative peptide in the treatment of cancer.
[0010] As a result, all the above-mentioned problems have made it necessary to make an innovation in the relevant technical field.
[0011] Objects of the Invention
[0012] The present invention relates to a method of using peptide inhibitors in the treatment of cancer in order to overcome the above-mentioned disadvantages and to bring new advantages to the relevant technical field.
[0013] One object of the invention relates to a method of using a protein-protein interaction inhibitor in the treatment of cancer, wherein the peptide sequence targeting and disrupting the Bag- IS / C-Raf interaction originates from the natural C-Raf sequence and binds to the Bag-1 S protein to disrupt the complex.
[0014] Another object of the invention relates to a method of using peptide inhibitors, namely Pl and its derivative peptide inhibitors, in the treatment of cancer types such as breast cancer and colon cancer in which the Bag-1 protein is overexpressed, and in the treatment of resistant Ras-mutant tumours in which dependence on the C-Raf protein develops.
[0015] Brief Description of the Invention
[0016] In order to fulfil all of the objects mentioned above and which will emerge from the detailed description below, the present invention is a method of using peptide inhibitors in cancer treatment targeting bag- 1 -mediated complexes, in particular the bag-l / c-raf complex. Accordingly, the novelty of the present invention comprises the use of piperidine to synthesise the peptide from rink amide resin as a solid support using a fmoc-based solidphase peptide synthesis approach to remove the firnoc protecting groups; the attachment of the amino acids using N,N-disopropylcarbodiimide and ethyl-2-cyano-2-(hydroxyimino) acetate for one cycle time; cleavage of protecting groups, treatment of peptides with 3 mL to 10 mL, preferably 5 mL of cleavage cocktail; precipitation of the peptide by centrifugation using chilled ether; C18 AdvanceBio Peptite Plus 2 at 214 and 280 nm. 7-Micron column at 214 and 280 nm; analysis of crude peptide using RP-HPLC; HPLC with a flow rate of 0.2 mL / min at 214 nm and 280 nm wavelengths with the help of UV detector using C-18 column. 2 mL / min; purification of the analysed peptides by HPLC using RPC ID column suitable for the system and peptide purification with the help of UV detector at 214 nm and 280 nm wavelengths with a flow rate of 1.8 mL / min to 2.5 mL / min, preferably 2 mL / min; linear gradient from 5% to 80% in 25 min to 40 min, preferably in 30 min, TF A / Acetonitrile from 10% to 40%, preferably in 0. 25%; lyophilisation of the peptide by removing the solvent under vacuum; lyophilisation of the peptide by adding a suitable solvent such as water or PBS to the lyophilised peptide; subsequent addition of the solubilised peptide directly to the media in which the cancer cell grows to affect the proliferation of the cancer cell. Thus, a peptide sequence that targets and disrupts the Bag-lS / C-Raf interaction, a protein-protein interaction inhibitor that originates from the natural C-Raf sequence and binds to the Bag- 1S protein and disrupts the complex can be used in cancer treatment. In addition, it can be used in the treatment of cancer types such as breast cancer and colon cancer in which Bag-1 protein is overexpressed and in the treatment of resistant Ras-mutant tumours in which dependence on C-Raf protein develops. Here, peptide inhibitors are Pl and derivative peptide inhibitors. In other words, the method of use of peptide inhibitors in which the cell permeable function is provided to the Pl peptide and the cell permeable P2 peptide, which is a derivative of Pl as a second peptide, is used in the treatment of cancer types and resistant Ras-mutant tumours. However, in a Pl as a peptide inhibitor, the liposome derivative can be taken into the cell in different ways and the effect can be achieved.
[0017] P2 peptide, which is a derivative of Pl, is cell permeable and Pl and liposome derivatives can be taken into the cell by different means and the effect can be achieved.
[0018] The basis of the invention is Pl and its derivative peptide inhibitors.
[0019] A possible embodiment of the invention is characterised in that the rink amide resin has a loading capacity of 0.70 mmol / g. Thus, it is possible to remove fmoc protection groups from the rink amide resin as desired.
[0020] A possible embodiment of the invention is characterised in that the heterocyclic amine organic compound piperidine is present in N,N-dimethylformamide in a loading capacity of 10% to 30%, preferably 20% by volume. Thus, it is possible to remove the fmoc protecting groups with piperidine as desired. A possible embodiment of the invention is characterised in that the molarity of the amino acids is 0.1 mol / L to 0.4 mol / L, preferably 0.2 mol / L. Thus, it can be ensured that the amino acids can be bound in the desired manner.
[0021] A possible embodiment of the invention is characterised in that the cycle time is between 2 and 10 minutes, preferably 4 minutes. Thus, it is ensured that the amino acids can be bound in the desired manner at the determined cycle time.
[0022] A possible embodiment of the invention is characterised in that the cleavage of the peptides is provided at 30°C to 45°C, preferably 37°C, for a period of 30 to 45 minutes. Thus, the peptides can be processed in the desired manner.
[0023] A feature of a possible embodiment of the invention is that the precipitation of the peptide by centrifugation is provided at 3000 rpm to 4500 rpm, preferably at 3500 rpm, for a period of 2 minutes to 10 minutes, preferably 5 minutes. Thus, the desired precipitation of the peptide can be achieved.
[0024] Brief Description of the Figures
[0025] Figure 1 shows a flowchart of a method of using peptide inhibitors in the treatment of cancer according to the invention.
[0026] Detailed Description of the Invention
[0027] In this detailed description, the method of use of peptide inhibitors in the treatment of cancer according to the invention is described only by means of non-limiting examples for a better understanding of the subject matter.
[0028] Figure 1 shows the flowchart of the method of using peptide inhibitors in the treatment of cancer.
[0029] The method of using peptide inhibitors in cancer treatment (10) targets bag- 1 -mediated complexes, in particular the bag-l / c-raf complex. The process steps in the flowchart of the method of use of peptide inhibitors (10) given in Figure 1 are given below; • Firstly, the peptide (12) is synthesised from rink amide resin (14) as a solid support using the fmoc-based solid phase peptide synthesis approach (18), and the fmoc protection groups are removed by the use of piperidine (16).
[0030] • Then the binding of amino acids (20), N,N-disopropylcarbodiimide (DIC) and ethyl-2- cyano-2-(hydroxyimino) acetate (22) is carried out in a cycle time (1 min for deprotection at 50 °C, 1 min for washing and 2 min for binding) using (24).
[0031] • Subsequently, treatment of the peptides by cleavage of the protecting groups (26) with 3 mL to 10 mL, preferably 5 mL of cleavage cocktail (TFA / H20 / TIS, 95 / 2.5 / 2.5, v / v / v) (28);
[0032] • Then precipitation of the peptide (34) by centrifugation (32) followed by centrifugation (30) using chilled ether (34).
[0033] • The crude peptide is then analysed (36) using RP-HPLC with a C18 AdvanceBio Peptite Plus 2.1 x 150 mm 2.7-Micron column at 214 and 280 nm.
[0034] • Then, using the C-18 column, the peptides synthesised in HPLC with a flow rate of 0.2 mL / min at 214 nm and 280 nm wavelengths with the help of UV detector (38) are analysed.
[0035] • The analysed peptides (36) (38) were then purified (40) (Solution A; 0.05% TFA / H2O (v / v) and Solution B; 0.25% TF A / Acetonitrile (v / v)) at 214 nm and 280nm wavelengths with the help of UV detector using RPC 250x10mm ID column suitable for the system and peptide purification (Solution A; 0.05% TFA / H2O (v / v) and Solution B; 0.25% TF A / Acetonitrile (v / v)).
[0036] • Peptides are then analysed by HPLC with a linear gradient (gradient) of 5% to 80% in 25 minutes to 40 minutes, preferably in 30 minutes, and TF A / Acetonitrile of 10% to 40%, preferably 0.25% (42).
[0037] • The peptide is then lyophilised by removing the solvent under vacuum (44).
[0038] • The lyophilised peptide is then dissolved by adding a suitable solvent such as water or PBS (46).
[0039] • Subsequently, the solubilised peptide is added directly to the media in which the cancer cell grows to affect the proliferation of the cancer cell (48). Said peptide can be administered to the patient orally, intravenously, intramuscularly, intraperitoneally, intraperitoneally, intradermally or directly into the tumour tissue by injection.
[0040] Furthermore, the rink amide resin (14) of the invention has a loading capacity of 0.70 mmol / g. Piperidine (16) is a heterocyclic amine organic compound in N,N- dimethylformamide in a volume ratio of 10% to 30%, preferably 20%. The molarity of the amino acids (20) is 0.1 mol / L to 0.4 mol / L, preferably 0.2 mol / L. The cycle time (22) of the invention is from 2 to 10 minutes, preferably 4 minutes. Furthermore, the cleavage of the peptides (26) is carried out at 30°C to 45°C, preferably at 37°C, for a period of 30 to 45 minutes. Precipitation (34) of the peptide by centrifugation (32) is also provided at 3000 rpm to 4500 rpm, preferably at 3500 rpm, for a period of 2 minutes to 10 minutes, preferably 5 minutes.
[0041] With regard to the method (10) for the use of peptide inhibitors in the treatment of cancer, the subject matter of the invention;
[0042] • First of all, the cytosolic small isoform of the anti-apoptotic Bag-1 protein and the serinethreonine protein kinase C-Raf protein involved in the MAPK cascade are cloned by molecular cloning approach as the whole sequence.
[0043] • Bag-1 S and C-Raf proteins were then produced in mammalian cell culture (HEK293T) and purified using polyhistidine and flag tags, respectively, which were added by cloning.
[0044] • The purified Bag-1 S is then immobilised on PVDF membrane and exposed to in vitro binding with C-Raf.
[0045] • Subsequently, limited tryptic cutting is applied following binding.
[0046] • The remaining C-Raf peptide fragment bound to Bag- IS is then obtained by acidic elution, enriched and analysed by mass spectrometry.
[0047] • Subsequently, the location of the identified fragment is evaluated on the C-Raf kinase domain structure and a shorter form is synthesised by the solid phase.
[0048] As a possible embodiment of the invention, the synthesis step of the peptide does not differ from the known solid phase peptide synthesis. However, the peptide sequence synthesised by the method of using peptide inhibitors (10) has anti-cancer properties. Possible embodiments of the invention are the use of a vector encoding said polypeptide, the use of a host cell containing said vector, the use of said polypeptide in the production of a drug for the treatment of cancer in patients, the use of a method of binding said polypeptide to molecular chaperones and the use of a pharmaceutical composition containing said polypeptide. With the method (10) of using the peptide inhibitors according to the invention, the following features are provided;
[0049] • In the method of using the peptide inhibitors (10) of the invention, the polypeptide sequences in the naturally occurring C-Raf protein with anti-cancer properties are the peptide Pl and the derivative of Pl is the peptide P2. Pl is “DNNPFSFQSDVYSYGIVL” and Pl derivative P2 is “GRKKRRQRRRPQDNNPFSFQSDVYSYGIVL”. Pl and P2 can be used individually or by deletion, insertion and / or single amino acid substitution or post-translational modifications, functionally active derivatives with the same anti-cancer activity can be obtained.
[0050] • In one embodiment of the invention, said polypetide sequence can be synthetically synthesised by means such as solid phase peptide synthesis and then purified by liquid chromatography.
[0051] • In another embodiment of the invention, the nucleotide sequence* encoding said peptide can be cloned into a vector and then transferred to a host organism (eukaryotic or prokaryotic) by transfection or transformation and produced recombinantly.
[0052] • In another embodiment of the invention, said polypeptide can be synthesised as an mRNA sequence encoding said peptide.
[0053] • The vector carrying the nucleotide sequence encoding the relevant polypeptide of the invention is available for integration into a host organism genome by stable transfection.
[0054] • In the invention, the recombinantly produced peptide can carry affinity labels at the N- or C- end for use in purification processes.
[0055] • In the invention, the recombinantly produced peptide can be purified by chromatographic means, i.e. affinity, ion exchange, etc.
[0056] • The peptide produced according to the invention can be distinguished according to its ability to bind to the Bag-1 protein by means of methods known in the literature, i.e. SPR, binding test through a labelled antibody, tests using covalent binding agents, etc.
[0057] • In the invention, the polypeptide obtained can be coated with liposome-derived carriers or nanoparticles to increase its cell permeability and / or to target certain types and characteristics of cancer cells.
[0058] • In the invention, the mRNA sequence* * encoding said polypeptide sequence can be used for therapeutic purposes. • The polypeptide obtained according to the invention is associated with a reduction in cancer cell survival. This effect can be studied in tumour cells or in tumour tissue of a patient.
[0059] • In the invention, said peptide can be used in cancer treatment alone or simultaneously or sequentially with another inhibitor in order to increase the anti-cancer effect.
[0060] • In the invention, said polypetite can be used in a therapeutically effective dose, in a solvent or diluent that can be used to provide chemical advantages such as increasing stability and / or solubility, or in different solution combinations.
[0061] • In the invention, said polypeptide can be administered to the patient orally, intravenously, intramuscularly, intraperitoneally, intradermally or by injection directly into the tumour tissue.
[0062] • In the invention, said polypeptide may be used alone or in combination with another active ingredient as an active ingredient in a drug formulation.
[0063] The process of synthesising peptide inhibitors according to the method of use of peptide inhibitors in cancer treatment (10) of the invention is given below;
[0064] • Firstly, the peptide is synthesised from rink amide resin as a solid support using firnoc- based solid phase peptide synthesis approach, and the fmoc protection groups are removed by the use of piperidine.
[0065] • Then the binding of amino acids is carried out using N,N-disopropylcarbodiimide (DIC) and ethyl-2-cyano-2-(hydroxyimino) acetate in a cycle time (1 min for deprotection, 1 min for washing and 2 min for binding at 50 °C).
[0066] • The cleavage of the protection groups is then performed by treating the peptides with 3 mL to 10 mL, preferably 5 mL of cleavage cocktail (TFA / H20 / TIS, 95 / 2.5 / 2.5 / 2.5, v / v / v).
[0067] • The peptide is then precipitated by centrifugation using chilled ether.
[0068] • The crude peptide is then analysed by RP-HPLC with a Cl 8 AdvanceBio Peptite Plus 2.1 x 150 mm 2.7-Micron column at 214 and 280 nm.
[0069] • Then, using the C-18 column, the peptides synthesised in HPLC with a flow rate of 0.2 mL / min at 214 nm and 280 nm wavelengths with the help of UV detector are analysed.
[0070] • The analysed peptides were then purified with a flow rate of 1.8 mL / min to 2.5 mL / min at 214 nm and 280 nm wavelengths, preferably 2 mL / min (Solution A; 0.05% TFA / H2O (v / v) and Solution B; 0.25% TF A / Acetonitrile (v / v)) with the help of UV detector using RPC 250x10mm ID column suitable for the system and peptide purification. • The peptides are then analysed by HPLC with a linear gradient (gradient) of 5% to 80% in 25 minutes to 40 minutes, preferably in 30 minutes, and TF A / Acetonitrile of 10% to 40%, preferably 0.25%.
[0071] • The peptide is then lyophilised by removing the solvent under vacuum.
[0072] • The lyophilised peptide is then dissolved by adding a suitable solvent such as water or PBS.
[0073] The Reference Numbers Given in the Figures
[0074] 10 Method of using peptide inhibitors
[0075] 12 Peptide
[0076] 14 Rink amid resin
[0077] 16 Piperidine
[0078] 18 Removal of fmoc protection groups
[0079] 20 Amino acids
[0080] 22 Cycle duration
[0081] 24 Performing the binding process
[0082] 26 Cleavage of protection groups
[0083] 28 Processing of peptides
[0084] 30 Use of chilled ether
[0085] 32 Centrifugation
[0086] 34 Precipitation of the peptide
[0087] 36 Analysis of the crude peptide
[0088] 38 Analysis of peptides
[0089] 40 Purification
[0090] 42 Analysis of peptides
[0091] 44 Lyophilisation of the peptide
[0092] 46 Solubilisation of the peptide
[0093] 48 Affecting cancer cell proliferation
Claims
CLAIMS1. A method for the use of peptide inhibitors in cancer treatment targeting bag- 1 -mediated complexes, in particular bag-l / c-raf complexes, characterised by comprising the process steps; removal of fmoc protecting groups by the use of piperidine (12) for the synthesis of the peptide (14) from rink amide resin (16) as a solid support using an fmoc-based solid phase peptide synthesis approach (18);- binding (24) of amino acids (20) using N,N-disopropylcarbodiimide and ethyl-2-cyano- 2-(hydroxyimino) acetate in one cycle duration (22); processing of peptides (28) by cleavage of protection groups (26), with 3 mL to 10 mL, preferably 5 mL of cleavage cocktail; precipitation of the peptide (30) by centrifugation (32) using chilled ether (34); analysis of the crude peptide using RP-HPLC with a C18 AdvanceBio Peptite Plus 2.7- Micron column at 214 and 280 nm (36); analysis of synthesised peptides in HPLC with a flow rate of 0.2 mL / min at 214 nm and 280 nm wavelengths with the help of UV detector using C-18 column (38); purification (40) of the analysed peptides (36) (38), using an RPC ID column suitable for the system and peptide purification, with the help of a UV detector at 214 nm and 280 nm wavelengths with a flow rate of 1.8 mL / min to 2.5 mL / min, preferably 2 mL / min; analysis of peptides (42) by HPLC with a linear gradient from 5 % to 80 % in 25 min to 40 min, preferably in 30 min, and TF A / Acetonitrile from 10 % to 40 %, preferably in 0.25 %; lyophilisation of the peptide by removal of the solvent under vacuum (44); solubilisation of the lyophilised peptide by adding a suitable solvent such as water or PBS (46);- then adding the solubilised peptide directly to the media in which the cancer cell grows, affecting the proliferation of the cancer cell (48).
2. A method of using peptide inhibitors in cancer treatment according to claim 1, characterised by the rink amide resin (14) having a loading capacity of 0.70 mmol / g.
3. A method of using peptide inhibitors in cancer treatment according to any one of the preceding claims, characterised in that the heterocyclic amine organic compoundpiperidine (16) is present in N,N-dimethylformamide in a proportion of 10% to 30%, preferably 20% by volume.
4. A method of using peptide inhibitors in cancer treatment according to any one of the preceding claims, characterised in that the molarity of the amino acids (20) is 0.1 mol / L to 0.4 mol / L, preferably 0.2 mol / L.
5. A method of using peptide inhibitors in cancer treatment according to any one of the preceding claims, characterised in that the cycle time (22) is between 2 and 10 minutes, preferably 4 minutes.
6. A method of using peptide inhibitors in cancer treatment according to any one of the preceding claims, characterised by the cleavage of peptides (26) at 30°C to 45°C, preferably at 37°C, for a period of 30 to 45 min.
7. A method of using peptide inhibitors in cancer treatment according to any one of the preceding claims, characterised by precipitation of the peptide (34) by centrifugation (32) at 3000 rpm to 4500 rpm, preferably at 3500 rpm and from 2 min to 10 min, preferably 5 min.